Sperm immobilizing activity of a synthetic bioactive peptide 20-44 of 37-kDa cationic antimicrobial protein (CAP37) of human neutrophils

We have previously reported that human sperm coincubated with human peripheral blood neutrophils in the presence of complement (C)-fixing antisperm antibody (ASA)-positive sera are rapidly internalized and degraded within the neutrophil phagolysosome. However, the mechanism by which motile sperm are processed within the phagolysosome is unknown. Various spermicidal/antimicrobial proteins contained in azurophilic granules that can be secreted into the phagolysosome may play a role in sperm disposal. In this study, we examined the expression of a 37-kDa cationic antimicrobial protein (CAP37) during sperm phagocytosis and the effect of its synthetic bioactive fragment, Peptide 20-44 (P20-44), on sperm motility, acrosomal integrity, and mitochondrial functionality. CAP37 expression by neutrophils undergoing ASA- and C-dependent sperm phagocytosis was increased as measured by flow cytometry. Exposure of motile sperm to a cationic P20-44, the bioactive antimicrobial fragment of CAP37, resulted in the loss of sperm motility without disruption of the acrosomal membrane. The sperm immobilizing activity (SIA) of P20-44 was modulated by the length of incubation, the concentration of the peptide, and the pH of the assay medium. SIA induced by P20-44 was partially reversible and was unaffected by the presence of anionic heparin or seminal plasma. Similar to the antimicrobial activity of P20-44, the SIA was also dependent on the presence of a disulfide bond between cysteine residues at positions 26 and 42 and was inhibited by Lipid A. However, the mechanism of action of P20-44 on sperm is not totally dependent on the molecule's cationicity, because five other cationic antimicrobial peptides had no detectable effect on sperm viability. Thus, the mechanism of action of P20-44 on human sperm is different from its cationic antibactericidal effect. These findings established that motile human sperm are sensitive to CAP37 or its synthetic bioactive peptide and suggested that this protein could play a role in neutrophil-mediated immune destruction of sperm in the female genital tract. P20-44 of CAP37 may be useful in investigating the regulation of human sperm motility and to construct "hybrid peptides" with enhanced potency as a component of vaginal contraceptive that could doubly be effective by killing infectious agents and inhibiting sperm transport.     

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.     

Primary myxoid leiomyoma of the liver

We describe a case of hepatic leiomyoma in a 41-year-old woman who complained of abdominal discomfort due to a right upper quadrant abdominal mass. A computed tomographic scan revealed a large hepatic mass with unusual cystic change. Light and electron microscopic findings and an immunohistochemical study demonstrated diffuse and scattered proliferation of smooth muscle cells in a myxoid matrix. In contrast to previously reported cases of primary leiomyoma of the liver, the present tumor developed in a patient without an immunosuppressive condition. To the best of our knowledge, this is the first reported case of primary "myxoid" leiomyoma of the liver.     

Integrated measurements of short-lived 222Rn progeny by rotating filters

The dependence of the risk from inhalation of radon progeny on their disequilibrium suggests that the measurements of the time-integrated concentrations of each of the short-lived radon progeny are necessary for complete risk estimations. This paper presents a method that, in principle, allows the determination of the integrated specific volume activities in air of each of the radionuclides 218Po, 214Pb, 214Bi, 212Pb, and 212Bi. The method employs thermoluminescence detectors positioned around uniformly rotating filters. Two prototypes that are suitable for practical applications are described and mathematical expressions for data processing are given. Experiments with these "rotating filter dosimeters" were conducted in atmospheres radiologically dominated by 222Rn progeny. The comparison between the results obtained by the proposed method and those given by simultaneously conducted series of instantaneous grab-sampling measurements support the conclusion that the method works for 222Rn progeny. The method can be experimentally extended for 220Rn progeny as well as for unattached fractions.     

Mice lacking E-selectin show normal numbers of rolling leukocytes but reduced leukocyte stable arrest on cytokine-activated microvascular endothelium

OBJECTIVE: Previous work indicated that E-selectin mediates transient interactions between leukocytes and cytokine-activated endothelium in vitro. Here we examine the role of E-selectin in blood leukocyte interactions with microvascular endothelium in vivo. METHODS: E-selectin-deficient (E-/-) mice were produced by gene targeting. The effect of this null mutation on leukocyte-endothelial interactions was determined by intravital microscopy before and 4 to 5 hours after local administration of the proinflammatory cytokine tumor necrosis factor alpha (TNF alpha) in dermal microvessels with low blood flow (dorsal skin-fold chambers, intact ear skin), and after endotoxin activation in exteriorized mesenteric microvessels with higher blood flow. RESULTS: E-/- mice were viable, fertile with normal circulating leukocyte and platelet profiles. Approximately 60% of circulating leukocytes rolled in dermal microvessels of both normal (E+/+) and E-/- mice without inflammatory stimulation. After local administration of TNF alpha, rolling increased modestly and equivalently in both genotypes. The main effect of TNF alpha was a dramatic increase in leukocyte stable adhesion and, unlike rolling, this manifestation of endothelial activation was significantly reduced in E-/- animals. This reflected fewer dermal microvessels supporting higher adhesion densities in E-/- mice, and a similar trend was observed in mesenteric microvessels. CONCLUSIONS: E-selectin plays a previously unappreciated role in facilitating and/or mediating stable adhesion of leukocytes to inflamed microvascular endothelium.     

Loss of heterozygosity on the long arm of human chromosome 7 in sporadic renal cell carcinomas

Cytogenetic and molecular analysis of DNA sequences with highly polymorphic microsatellite markers have implicated allele loss in several chromosomal regions including 3p, 6p, 6q, 8p, 9p, 9q, 11p and 14q in the pathogenesis of sporadic renal cell carcinomas (RCCs). Deletions involving the long arm of chromosome 7 have not been described in RCCs although they have been seen in several other tumor types. However, there have been no detailed analysis of loss of heterozygosity (LOH) of 7q sequences in sporadic RCCs. We therefore studied LOH for DNA sequences on 7q with 10 highly polymorphic markers in 92 matched normal/tumor samples representing sporadic RCCs including papillary, nonpapillary, and oncocytomas in order to determine whether allelic loss could be detected in a tumor type with no visible 7q rearrangements at the cytogenetic level. We found chromosome 7q allele loss in 59 of 92 cases (64%) involving one, two, or more microsatellite markers. The most common allele loss included loci D7S522 (24%) and D7S649 (30%) at 7q31.1-31.2, a region that contains one of the common fragile sites, FRA7G. By comparative multiplex PCR analysis, we detected a homozygous deletion of one marker in the 7q 31.1-31.2 region in one tumor, RC21. These results support the idea that a tumor suppressor gene in 7q31 is involved in the pathogenesis of sporadic renal cell carcinomas.     

Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.